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. 2016 Aug 25;291(43):22442–22459. doi: 10.1074/jbc.M116.754069

FIGURE 3.

FIGURE 3.

ZC3H14 specifically regulates the ATP5G1 transcript. A, ATP5G1, ATP5G2, and ATP5G3 transcripts are transcribed from three separate genomic loci on chromosomes (Chr.) 17, 12, and 2, respectively. The ATP5G1, -2, and -3 genes and their corresponding mRNAs have varying lengths (reported in base pairs (bp) or nucleotides (nt); thin bars = introns, thicker bands = UTRs, and boxes = coding regions) and encode distinct protein products (P1, P2, and P3). ATP5G2 is alternatively spliced to form two distinct mRNAs and subsequent protein products, P2a and P2b. The encoded protein products contain identical C termini with variable N-terminal mitochondrial targeting peptides (red, with the number of amino acids indicated) that are cleaved (red lightning bolt) upon import into the mitochondria. The resulting mature protein products (dark gray) are completely identical in amino acid sequence (76 amino acids). B, total RNA isolated from MCF-7 cells was used for qRT-PCR analysis with primers specific to each of the ATP5G mRNAs. The relative value of each ATP5G mRNA was calculated by 2∧−Ct and is reported as relative units. C, MCF-7 cells transfected with Scramble control or ZC3H14 siRNA were subjected to RNA isolation and qRT-PCR analysis with primers specific to all three ATP5G mRNAs as well as the control transcript, RPLP0. Values are set to 1.0 for siScramble and normalized to RPLP0. Knockdown of ZC3H14 results in a specific and robust decrease in ATP5G1 steady-state mRNA levels. D, endogenous nuclear isoforms of ZC3H14 were immunoprecipitated from MCF-7 cells using either ZC3H14 antibody-bound protein A/G beads or control rabbit pre-immune serum-coated beads. Proteins from the input (I), unbound (UB), and bound (B) fractions were resolved on an SDS-polyacrylamide gel and subjected to immunoblotting with ZC3H14 antibody. The nuclear ZC3H14 isoforms were detected in the ZC3H14-bound fraction but not the pre-immune bound fraction. E, RNA isolated from the ZC3H14 RNA-IP was subjected to qRT-PCR analyses with GAPDH, RPLP0, ATP5G1, ATP5G2, ATP5G3, and ZC3H14 primers. mRNA levels in the ZC3H14 bound fractions were normalized to input levels and then compared by fold-enrichment over pre-immune control. Significant enrichment of ATP5G1, ATP5G3, and ZC3H14 transcripts was observed with ZC3H14 IP. Values represent the mean ± S.E. for n = 3 independent experiments. **, ***, and **** represent p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001, respectively.